Stimulus secretion coupling in pancreatic beta-cells

在过去的几年里，我们的主要活动之一是开发一个全面的模型，用于从秒到分钟的时间尺度上的膜电位和钙的振荡。 这些导致胰岛素分泌的相应振荡。 该模型的基本假设是，更快的振荡（数十秒）源于钙离子通道的反馈，可能是钙激活钾（K（Ca））通道和ATP依赖性钾（K（ATP））通道，而较慢的振荡（5分钟）源于代谢振荡。 代谢振荡通过K（ATP）通道转换成电振荡。 因此，该模型由电振荡器（EO）和代谢振荡器（MO）组成，并且被称为双振荡器模型（DOM）。 在我们的模型中，MO是一个糖酵解振荡器，但如果代谢振荡发生在其他地方，如线粒体，系统的许多特征仍然成立。 K（ATP）通道具有临床意义，因为它们是用于治疗2型糖尿病的胰岛素刺激药物（例如磺酰脲类甲苯磺丁脲和格列本脲）的一线靶标。 严重的K（ATP）功能获得性突变是新生儿糖尿病的主要原因，而在全基因组关联研究（GWAS）中，中度功能获得性突变与较轻但更常见的疾病成人发作的2型糖尿病有关。 相反，K（ATP）的功能丧失突变是家族性高胰岛素血症的主要原因，家族性高胰岛素血症是一种在儿童中发现的遗传性疾病，其中β细胞持续电活性并在正常或低血糖时分泌胰岛素，导致危及生命的低血糖。 糖酵解振荡模型中的关键因素是磷酸果糖激酶-1（PFK 1）的活性通过其产物果糖-1，6-二磷酸（Fru 1，6-BP）的正反馈。 然而，一种相关的酶，磷酸果糖激酶-2（PFK 2）产生果糖-2，6-二磷酸（Fru 2，6-BP），其比Fru 1，6-BP更强烈地激活PFK 1。 PFK 2是一种特别有趣的分子，因为它是双功能酶（BIF 2）的一部分，该酶含有激酶及其相应的磷酸酶果糖-2，6-二磷酸酶（FBPase 2）。 我们在Satin实验室（密歇根大学）的合作者使用了一种新技术来过表达PFK 2或FBPase 2或BIF 2，其中包含激酶死亡或磷酸酶死亡部分。 我们发现，增加相对于磷酸酶的激酶活性增加的频率，降低缓慢的钙振荡的幅度，而增加磷酸酶的相对活性有相反的效果。 模型的模拟和分析表明，Fru 2，6-BP的增加通过降低PFK 1被其底物6-磷酸果糖激活的阈值来增加频率。 然而，PFK 2本身不能驱动振荡，因为它不能从其产品接收正反馈。 实验观察结果与DOM预测的一致性进一步支持了该模型，特别是支持代谢振荡起源于糖酵解而不是线粒体的假设;很难看出糖酵解的特定修饰如何对线粒体振荡器产生所观察到的影响。 这项工作在参考文献1中有描述。 在更理论的层面上，我们在对各种细胞类型中发现的爆发振荡进行分类并将β细胞与其他形式联系起来的长期目标方面取得了重要进展。 这在我们关于神经元和内分泌细胞的数学建模的项目报告中有详细描述。 正在进行的其他研究包括测量和模拟K（ATP）通道电导振荡，建模敲除离子通道Trpm 5以选择性抑制快速电振荡的效果，并将DOM扩展到分泌胰高血糖素的胰腺α细胞。

One of our main activities over the last few years has been the development of a comprehensive model for oscillations of membrane potential and calcium on time scales ranging from seconds to minutes. These lead to corresponding oscillations of insulin secretion. The basic hypothesis of the model is that the faster oscillations (tens of seconds) stem from feedback of calcium onto ion channels, likely calcium-activated potassium (K(Ca)) channels and ATP-dependent potassium (K(ATP)) channels, whereas the slower oscillations (five minutes) stem from oscillations in metabolism. The metabolic oscillations are transduced into electrical oscillations via the K(ATP) channels. The model thus consists of an electrical oscillator (EO) and a metabolic oscillator (MO) and is referred to as the Dual Oscillator Model (DOM). In our model, the MO is a glycolytic oscillator, but many of the features of the system would still hold if the metabolic oscillation arose elsewhere, such as the mitochondria. K(ATP) channels are of clinical significance as they are a first-line target of insulin-stimulating drugs, such as the sulfonylureas tolbutamide and glyburide, used in the treatment of Type 2 Diabetes. Severe gain-of-function mutations of K(ATP) are a major cause of neo-natal diabetes mellitus, whereas moderate gain-of-function mutations have been linked in genome-wide association studies (GWAS) to the milder but more common disease, adult-onset type 2 diabetes. Conversely, loss-of-function mutations of K(ATP) are a major cause of familial hyperinsulinism, a hereditary disease found in children in which beta cells are persistently electrically active and secrete insulin in the face of normal or low glucose, causing life-threatening hypoglycemia. The key element in our model for glycolytic oscillations is the positive feedback on the activity of phosphofructokinase-1 (PFK1) by its product fructose-1,6-bisphosphate (Fru1,6-BP). However, a related enzyme, phosphofructokinase-2 (PFK2) produces fructose-2,6-bisphosphate (Fru2,6-BP), which activates PFK1 even more strongly than Fru1,6-BP. PFK2 is a particularly interesting molecule in that it is part of a bifunctional enzyme (BIF2) that contains both the kinase and its corresponding phosphatase, fructose-2,6-bisphosphatase (FBPase2). Our collaborators in the Satin lab (University of Michigan) used a novel technique to overexpress either PFK2 or FBPase2 or BIF2 with either kinase-dead or phosphatase-dead moieties. We found that increasing the activity of the kinase relative to the phosphatase increases the frequency and reduces the amplitude of slow calcium oscillations whereas increasing the relative activity of the phosphatase had the opposite effect. Simulations and analysis of the model showed that increased Fru2,6-BP increases frequency by lowering the threshold for PFK1 activation by its substrate, fructrose-6-phosphate. PFK2, however, is not able to drive oscillations by itself since it does not receive positive feedback from its product. The agreement of the experimental observations with the predictions of the DOM lends further support to the model, in particular supporting the hypothesis that the metabolic oscillations originate in glycolysis rather than, say, the mitochondria; it is difficult to see how a specific modification of glycolysis would have the observed effects on a mitochondrial oscillator. This work is described in Ref. # 1. On a more theoretical level, we have made important progress in our long-term goal of classifying bursting oscillations found in a variety of cell types and relating the beta-cell variety to other forms. This is described in detail in our project report on Mathematical Modeling of Neurons and Endocrine Cells. Other studies in progress include measurements and simulations of K(ATP) channel conductance oscillations, modeling the effect of knocking out the ion channel Trpm5 to selectively suppress fast electrical oscillations, and extending the DOM to pancreatic alpha cells that secrete glucagon.